US5143844A - Construction of an igg binding protein to facilitate downstream processing using protein engineering - Google Patents

Construction of an igg binding protein to facilitate downstream processing using protein engineering Download PDF

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US5143844A
US5143844A US07/594,564 US59456490A US5143844A US 5143844 A US5143844 A US 5143844A US 59456490 A US59456490 A US 59456490A US 5143844 A US5143844 A US 5143844A
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fragment
codon
protein
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Lars Abrahmsen
Tomas Moks
Bjorn Nilsson
Mathias Uhlen
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Pfizer Health AB
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/705—Fusion polypeptide containing domain for protein-protein interaction containing a protein-A fusion
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates to a recombinant DNA fragment coding for an immunoglobulin G (hereinafter called IgG) binding domain related to staphylococcal protein A, to DNA sequences comprising such fragments and to a process for cleavage of a fused protein expressed by using such fragment or sequence.
  • IgG immunoglobulin G
  • the invention also relates to plasmid vectors and bacterial cells harboring such recombinant DNA fragments or sequences. Basically, the present invention relates to an improved system for producing and purifying foreign proteins expressed in bacteria.
  • Gene fusion techniques have been used in recombinant DNA technology to monitor the transient expression from a gene or to facilitate the downstream processing.
  • any gene product can be purified as a fusion protein to protein A and can thus be purified in a single step using IgG affinity chromatography.
  • IGF-I human insulin-like Growth Factor I
  • the hybrid protein expressed could be recovered in high yield from the growth medium of Staphylococcus aureus.
  • EE-IGF-I a gene product consisting of divalent protein A fused to IGF-I was secreted from the E. coli cell by a method described in our Swedish patent application (the disclosure of which is incorporated herein by reference; filed simultaneously herewith).
  • IGF-I the method suggested by us to be used is hydroxylamine cleavage in Asn-Gly dipeptide sequences. The method mostly used is otherwise CNBr cleavage specific for Met. The choice of method is dependent on if the amino acid(s) sensitive for the chemical is present in the product or not.
  • IGF-I has an internal methionine and IGF-II does not.
  • Protein A has, however, 3 internal methionines in the IgG binding region and 5 Asn-Gly in the IgG binding region of protein A. This makes the second passage through the column irrelevant as the protein A pieces released from the cleavage will not bind to the IgG.
  • the main object of this invention is to provide a solution to these problems by adapting an IgG binding domain so that no Met and optionally no Asn-Gly is present in the sequence.
  • two non-palindronic AccI sites are preferably introduced in the fragment to be able to polymerize the IgG binding domain to any number of IgG binding domains.
  • the invention provides for a recombinant DNA fragment (abbreviated Z in this disclosure) coding for an immunoglobulin G binding domain related to staphylococcal protein A, such fragment being characterized in that the methionine codon of said fragment has been replaced by a codon of another amino acid residue enabling expression of a methionine-free protein. It is preferred that the codon of said other amino acid residue is that of leucine.
  • the asparagine-methionine codons have been replaced by histidine-leucine codons.
  • the codon of amino acid residue No. 1 as defined by trypsin digestion of native protein A is preferably replaced by a valine codon, so as to give at the nucleotide level the sequence GTAGAC furnishing a non-palindromic Acc I site.
  • the glycine codon in the Asn-Gly constellation has been replaced by an alanine codon.
  • the Asp-Pro codons have suitably been modified to increase the acid stability of the peptide bond of the expressed protein, for example by replacing the aspartic acid codon with a glutamic acid codon.
  • a recombinant DNA sequence comprising at least two Z-fragments as defined above.
  • the number of such amalgamated Z-fragments is preferably within the range 2-15, and particularly within the range 2-10.
  • the invention also provides for a recombinant DNA sequence comprising the Z-fragment as defined above preceded by a signal sequence followed by a nucleotide sequence coding for the amino acid sequence: Ala Gln His Asp Glu Ala.
  • the invention also covers a recombinant DNA molecule comprising the recombinant DNA sequence as described above and fused at the 3' end thereof at DNA level, a production gene.
  • a production gene may be that of a somatomedin, examples of which are: growth hormones or factors, such as hGH (human growth hormone), IGF-I, IGF-II, NGF (Nerve Growth Factor), EGF (Epithermal Growth Factor) and PDGF (Platelet Derived Growth Factor).
  • the production gene may also be one coding for an interferon, interleukin-2, insulin, neuropeptide, gastrointestinal peptide etc. Specifically, the production gene is that of IGF-I or IGF-II.
  • the production gene may also code for a structural gene for an enzyme or parts thereof.
  • the N-terminal glycine codon is preceded by an asparagine codon to enable hydroxyl amino cleavage of the peptide bond to release the native protein, such as IGF-I.
  • the N-terminal codon is preceded by a methionine codon to enable cyanogen bromide cleavage of the peptide bond to release native protein, such as IGF-II.
  • a process for cleaving a fused protein expressed in a biological system by the recombinant DNA molecule as defined above is suitably performed by hydroxyl amine treatment when the N-terminal glycine codon is preceded by an asparagine codon.
  • the cleavage is preferably performed by cyanogen bromide treatment.
  • the invention covers a plasmid vector comprising the recombinant DNA molecule as described above.
  • the invention also extends to bacterial cells harboring the recombinant DNA-molecule defined above.
  • the molecule can be harbored in the chromosome of the bacterial cell but may also be contained in a plasmid vector.
  • the host cell is for example Gram negative and is particularly constituted by an E. coli.
  • FIG. 1 shows the organization of the coding region of the protein A gene.
  • S is the signal sequence
  • A-E are the IgG binding domains
  • X is the C-terminal region with no IgG binding activity of the encoding polypeptide
  • FIG. 2 shows a comparison of the different IgG binding regions.
  • the first line shows a suggested consensus amino acid sequence of the IgG binding regions.
  • the boxes show the stretches of amino acids involved in the two different alpha helixes.
  • the amino acids involved in the binding to IgG are underlined.
  • the amino acids in the different regions are shown by -for no change compared to the consensus codon, +for no amino acid change but a silent mutation and the letter for another amino acid for amino acid changes.
  • the amino acids are given in the one letter code;
  • FIG. 3 shows the nucleotide sequence of the sense strand of the synthezised Z-fragment.
  • the cleavage region is the stretch of amino acids needed for processing of the signal sequence.
  • the Z-region is the part of the Z-fragment coding for the IgG binding domain.
  • the amino acid changes are underlined.
  • the restriction enzyme recognition sequences for sites used in the Examples are shown;
  • FIG. 4 shows the nucleotide sequence of the ZZ-IGF-I encoded by the pZZ-IGF-I plasmid vector. The regions encoding the signal peptide, the cleavage region, the two Z-regions and IGF-I are shown as well as restriction sites relevant for the construction strategy;
  • FIG. 5 shows the strategy described in Examples section V.
  • the synthetic oligomers were cloned in M13 mp 18 (not shown in the figure) prior to the cloning of the Z-fragment (from Hind III to Eco RI) into pUC8.
  • AMP is the gene coding for the ⁇ -lactamase gene
  • ori is the origin of replication for E.coli
  • lac Z' is the gene coding for ⁇ -galactosidase alpha fragment
  • Z is the synthetic fragment
  • FIG. 6 shows the cloning strategy described in Examples section VI.
  • AMP is the gene coding for ⁇ -lactamase
  • S is the signal sequence
  • A-E are the IgG binding domains of protein A
  • ori is the origin of replication
  • Z is the synthetic fragment
  • IGF-I is the gene for IGF-I
  • F1 is the origin of replication from phage f1
  • lacZ is the gene for ⁇ -galactosidase
  • FIG. 7 shows the construction of pASZ1 and pASZ2 as, described in Examples sections III and IV.
  • AMP is the gene encoding the ⁇ -lactamase
  • F1 is the origin of replication for phage f1
  • S is the signal sequence
  • A-E are the IgG binding regions
  • ori is the origin of replication for E.coli and Z is the synthetic fragment;
  • FIG. 8 shows the strategy of the process to purify IGF-I using the method of unique Asn-Gly cleavage, as described in Examples section VII;
  • FIG. 9 shows the SDS gel electrophoresis of the proteins corresponding to the different steps in the process described in Examples section VII.
  • Lane 1 shows size markers in Kilo Daltons
  • lane 2 shows the hydrid protein after IgG affinity purification
  • lane 3 shows the result of hydroxylamine cleavage
  • lane 4 shows the flow through of an IgG sepharose gel of hydroxylamine digested hybridprotein
  • lane 5 is pure IGF-I (marker).
  • the bands corresponding to ZZ-IGF-I, ZZ and IGF-I are shown by arrows.
  • the fragment used to illustrate the invention was constructed in the following way:
  • Protein A consists of two distinct regions: The IgG binding region (A-E domains) and region X having no IgG binding activity (FIG. 1).
  • the IgG binding region consists of five homologous IgG binding domains which can be cleaved apart by trypsin treatment at the protein level.
  • the B-domain has been crystallized together with IgG and the structure determined by X-ray crystallography (Deisenhofer, J., Biochemistry. 20, 2361 (1981).
  • the five IgG binding domains consist of approximately 58 amino acids (E is shorter and D is longer) and the amino acid sequences of the regions are shown in FIG. 2.
  • the fragment is optimized to be synthesized at the nucleotide level to facilitate cloning to get expression
  • the fragment can be polymerized at the nucleotide level to get any number of IgG binding regions
  • the fragment is capable of being expressed in a genetic system adapted for expression and secretion.
  • the Asn-Gly dipeptide sequence is sensitive to hydroxylamine. As this sequence is kept intact in all five IgG binding domains of protein A and as this amino acid sequence is present in the middle of an alpha helix involved in the binding to IgG (FIG. 2) there is very little chance to be successful in any amino acid change.
  • the obvious choice to change the Asn to a Gln was analyzed by computer graphics (FRODO software, Alwin Jones, Biomedical Centre, Uppsala, Sweden) using the coordinates available from the Brookhaven Protein Data Bank (Bernstein, F.C. et al J.Mol.Biol., 112, 553-542 (1972) calculated from the X-ray crystallographic structure of protein A.
  • the fragment was synthesized at the at DNA level in 10 separate oligomers. To facilitate cloning of the fragment one HindIII site was designed in the 5' end and an EcoRI site in the 3' end. (The nucleotide sequence of the fragment is shown in FIG. 3).
  • a non-palindromic AccI site was introduced by changing the nucleotide sequence to GTAGAC in the code for the N-terminal end of the IgG binding region. In that way an Ala codon present in all regions is changed to a Val.
  • the fragment can be polymerized to any multiplicity thus altering the binding capacity of the translated product. It is not obvious if this amino acid substitution will interfere with the protein A function or not.
  • HB101 Boyer, H. W. et al J.Mol.Biol., 41, 459-572 (1969)
  • JM 83 Yanisch-Perron, C. et al Gene, 33, 103-119 (1985)
  • JM 103 Messing, J. et al Methods Enzymol., 101, 20-79 (1983)).
  • the strains are available at the Dept of Biochemistry and Biotechnology, Royal Insitute of Technology, Sweden.
  • the cloning vehicles used in the Examples were M13 mp 18 (Yanisch-Perron, C. et al Gene, 33, 103-119 (1985)) and pUC8 (Vieva, J. et al Gene 19, 259 (1982)).
  • the vector pHL33 is a vector derived from pEMBL19 (-) (Dente et al, Nucl.Acids Res., 11, 1645 (1983)) and pRIT4 (Nilsson, B. et al, EMBO J. 4, 1075 (1985)) constructed in the following way:
  • the plasmid pRIT4 was cleaved with Taq I and after fill-in reaction with Klenow polymerase, Not-I linkers were added. The reaction mixture was cleaved with EcoRI Not I and the fragment spanning over the protein A gene was isolated. This fragment was cloned into pEMBL19 (-), where the ClaI site previously had been linked to a NotI site, by cleaving that plasmid with NotI and EcoRI. This gives a vector containing a part of the protein A gene followed by the mp19 multirestriction enzyme linker.
  • the vector pEX4-IGF-I is pEX (Stanley, K. et al EMBO J. 3, 1429 (1984) having the synthetic IGF-I cloned in EcoRI to BamHI'.
  • the synthetic gene encoding IGF-I has been described by Elmblad, A. et al in Third European Congress on Biotechnology III, 287-296, Verlag Chemie, Weinheim (1984).
  • the plasmid vector pASZ2 has been deposited with the Deutsche Sammlung von Mikroorganismen (DSM), Gottingen, Federal Republic of Germany, under No. 3594 DSM and (the designated name pE*2 in the deposition document).
  • Transformation of E. coli K12 with plasmid DNA was performed exactly as described (Morrison, D. A., Methods in Enzymology, Academic Press 68, 326-331 (1979)). The transformants were selected in a conventional manner on plates (TBAB) containing 70 mg/l ampicillin.
  • Plasmid DNA was isolated as described by Birnboim, H. C. et al, Nucl. Acids Res. 7, 1513 (1979). Small scale preparations to screen a large number of transformants were made exactly as described by Kieser, T. Plasmid 12, 19-36 (1984).
  • Plasmid DNA to be used for Bal31 or S1 treatment were run on a Sepharose 6B gelfiltration in a 10 mM Tris, 1 mM EDTA and 500 mM NaCl-buffer. In this way DNA is separated from RNA.
  • Ligation directly in agarose gel was performed by running the electrophoresis in a Low Gel Temperature Agarose gel and after cutting out the band, the gel piece was melted by heating to 65° C. After a 10 fold dilution using Tris buffer (10 mM pH 7.4) ligation could be performed.
  • ELISA test Enzyme linked immunosorbent assay
  • the test makes use of a special microtiter plate (Titertek, Amstelstad, Netherlands) having no net charge.
  • the wells are coated with human IgG (Kabi AB, Sweden) in a coating buffer.
  • Test samples are added and protein A is bound to the Fc portions of the IgG adsorbed in the well. Protein A is then assayed by an anti-protein A (from rabbit) conjugated to ⁇ -galactosidase (from Pharmacia AB, Uppsala, Sweden).
  • the wells of a microtiter plate are filled with 75 ⁇ l of a solution of human IgG at 16 ng/ml in Coating Buffer and the plate is incubated at room temperature for at least 1 hour.
  • the wells are washed three times with 100 ⁇ l PBST and 50 ⁇ l of sample is added to each well. For quantitative determination 2 fold dilutions are made. After incubation for 1 hour the wells are washed 3 times with 100 ⁇ l PBST followed by addition of 50 ⁇ l anti-protein A- ⁇ -galactosidase (the amount of protein A binding capacity added to each well corresponds to the molar amount of IgG added to each well as detected by titration with protein A in excess).
  • SDS-polyacrylamide gel electrophoresis was performed exactly as described by Laemmli, O.K. Nature (London), 227, 680-685 (1970) using a 10-20% step gradient gel.
  • the derived DNA sequence was analyzed by a computer program and divided into 10 oligonucleotides varying in length from 41 to 45 nucleotides and with an overlap of 6 bp.
  • Synthesis was effected on a fully automated machine and the deprotected oligomers were purified by polyacrylamid electrophoresis (20% polyacrylamide, 7M Urea, 50 mM Tris-borate pH 8.3) followed by extraction into water and lyophilization.
  • oligonucleotides A1-A5 and B1-B5 were phosphorylated separately in 20 ⁇ l Kinase buffer (50 mM Tris-HCl pH 7.6, 10 mM MgCl 2 , 1 mM ATP. 10 mM DTT) 5 units of polynucleotide kinase was added and the mixtures were incubated for 45 minutes at 37° C.
  • Kinase buffer 50 mM Tris-HCl pH 7.6, 10 mM MgCl 2 , 1 mM ATP. 10 mM DTT
  • 5 ⁇ g of the vector M13mp18, replicative form was digested with the restriction enzymes EcoRI and HindIII.
  • the large fragment from the digestion was isolated from a low temperature agarose gel.
  • the agarose containing digested M13 mp18 was melted at 65° C. and 5 ⁇ l (0.5 ⁇ l, 0.1 p mole) was mixed with 0.5 p mole each of the phosphorylated oligomers A1-A5 and B1-B5 in 50 ⁇ l ligation buffer (66 mM Tris-HCl pH 7.6 50 mM MgCl 2 , 50 mM DTT, 1 mM ATP) heated to 90° C. and slowly cooled to room temperature during one hour. 10 units of T4 DNA ligase was added and the mixture was incubated overnight at 15° C.
  • 50 ⁇ l ligation buffer 66 mM Tris-HCl pH 7.6 50 mM MgCl 2 , 50 mM DTT, 1 mM ATP
  • E. coli JM 103 was transfected with the DNA thus obtained and grown overnight on 2 ⁇ YT plates containing x-gal and IPTG. 78 white plaques were transferred to a new 2 ⁇ YT plate and grown as colonies. Colony hybridization with 32 P labelled oligonucleotide B5 as probe gave one positive colony which was picked and grown in 15 ml 2 ⁇ YT with E. coli JM 103. The cells were spun down and the phages were recovered from the supernatant. The single stranded phage DNA was extracted and purified and was used as template for the sequencing reaction according to the dideoxy method. The M13mp18 containing the Z-fragment was designated M13-Z.
  • the plasmid vector PHL33 was cleaved with HindIII and EcoRI. The large fragment was isolated from a 1% agarose gel after electrophoresis. The fragment was ligated to an isolated Z-fragment (HindIII/EcoRI). After ligation the mixture was transformed to E. coli HB101 and the isolated vector PASZ1 (FIG. 7) has the synthetic Z-fragment cloned down stream from the signal sequence. In order to place the synthetic fragment directly after the signal sequence the pASZ1 vector was cleaved with FspI. After religation the mixture was transformed into E. coli HB101 and pASZ2 could be isolated.
  • the vector has the synthetic Z-fragment (FIG. 3) directly after the signal sequence of protein A.
  • the strain was inoculated to 15 ml TSB and after incubation in a shake flask for 12 h the cell suspension was centrifuged.
  • the cells were washed once in TE (5 ml) and was further resuspended in 5 ml TSB followed by sonication for 3 ⁇ 30 s (MSE sonicator, microtip, power 6). After sonication the mixture was centrifuged 10,000 xg for 10 minutes.
  • the single stranded DNA from M13-Z was annealed to the sequencing primer followed by treatment with Klenow and dNTPs.
  • the pUC 8-Z was digested with restriction enzyme AccI. After religation of the digestion mixture a new transformation of JM83 was made, followed by plasmid isolation from 12 transformants. Digestion with HindIII and EcoRI and analysis on agarose gel confirmed that 2 transformants carried the pUC 8 with an insert of two Z fragments (pUC 8-ZZ).
  • pUC18-ZZ was digested with FspI and EcoRI and the smallest fragment was isolated on LGT agarose.
  • the plasmid vector pHL33 was digested with FspI. The largest fragment (2273 bp) was isolated on LGT agarose.
  • the plasmid pEX4-IGF-I was digested with FspI and EcoRI. The small fragment spanning over the IGF-I gene into the AMP gene was isolated.
  • the three fragments from A, B and C were ligated together as described in Routing Methods and the ligation mixture was transformed into E. coli JM83.
  • Transformant selection was conducted using a LB agar medium containing 70 ⁇ g/ml of ampicillin. Isolation of the plasmid DNA and analysis with restriction enzymes confirmed that the transformants carried the plasmid pZZ-IGF-I.
  • the supernatant was passed through the column at a speed of 12 ml/h and the amount of IgG binding material was analyzed before and after it was run through the column.
  • the bound material was washed with TS supplemented with 0.05% Triton X-100 and then TS and finally with 0.05M ammonium acetate before elution with 1M acetic acid pH adjusted to 2.8 with ammonium acetate.
  • the 2 ml fractions were assayed for protein A content and the fractions were pooled and freeze dried.
  • the hybrid protein was cleaved with hydroxyl amine as described by Bornstein, et al, Methods Enzymol., 47, 132 (1977).
  • the dry material was dissolved in 2M hydroxylamine adjusted to pH 9 with lithium chloride and 0.2M Tris base. The cleavage was performed at 45° C. for 4 h. The cleavage reaction was stopped by lowering the pH to 7.0 with acetic acid and the material was desalted on a PD-10 column saturated with TS. The desalted fraction of 3.5 ml was passed through a 1 ml IgG-sepharose column to separate IGF-I from the ZZ polypeptide. This material was then desalted on a PD-10 column saturated with 0.2M acetic acid, and then freeze dried. The material was then analyzed on sodium dodecyl sulphate polyacrylamide gel electrophoresis. This process is shown in FIG. 8, and the SDS-polyacrylamide gel is shown in FIG. 9. The nucleotide sequence of the expressed fusion protein is shown in FIG. 4.
  • the ZZ fragment is functional in a process to purify IGF-I in respect to binding IgG to facilitate purification as well as harboring a unique Asn-Gly cleavage site to release native IGF-I and to enable the ZZ portion to bind to the IgG gel after a second passage through the gel.
  • IGF-I-purification is only one example of the usefulness of the invention and is not to be construed to limit the scope of the invention otherwise than as defined in the appended patent claims.
  • the Asp-Pro dipeptide constellation present in all protein A domains known to bind human IgG was designed to be changed to a Glu-Pro. This will make the normally acid labile Asp-Pro peptide bound resistant to acid treatment (like 70% Formic Acid at 42° for 12 h) making any other introduced Asp-Pro coding linker unique. This designed amino acid change (Asp to Glu) is not shown in Examples.

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WO1997029127A1 (en) * 1996-02-06 1997-08-14 Bionebraska, Inc. Recombinant preparation of calcitonin fragments and use thereof in the preparation of calcitonin and related analogs
US5863783A (en) * 1991-03-27 1999-01-26 Gist-Brocades, N.V. Cloning and expression of DNA molecules encoding arabinan-degrading enzymes of fungal origin
US5917026A (en) * 1996-02-05 1999-06-29 Loewenadler; Bjoern Fusion proteins of immunopotentiating activity
US5985599A (en) * 1986-05-29 1999-11-16 The Austin Research Institute FC receptor for immunoglobulin
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EP0230869B1 (en) 1992-09-02
AU600244B2 (en) 1990-08-09
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JPS62190087A (ja) 1987-08-20
AU6633186A (en) 1987-06-18
JPH0811069B2 (ja) 1996-02-07
DE3686646T2 (de) 1993-04-08
ATE80180T1 (de) 1992-09-15
CA1311430C (en) 1992-12-15
EP0230869A3 (en) 1988-08-03
GR3006178T3 (cs) 1993-06-21
DK600486A (da) 1987-06-14
SE8505922D0 (sv) 1985-12-13
DE3686646D1 (de) 1992-10-08
EP0230869A2 (en) 1987-08-05
DK600486D0 (da) 1986-12-12

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